JP6067020B2 - Nucleic acid sequence cleavage labeling method - Google Patents

Description

The present invention relates to a method for cleaving a nucleic acid sequence for labeling whether or not a target nucleic acid has been cleaved, and a probe set for determining pathophysiological cleavage used in the method for cleaving and labeling a nucleic acid sequence.

Known methods for visually locating specific nucleotide sequences and genes include fluorescence in situ hybridization (FISH method) and chromogenic in situ hybridization (CISH) method.

In the FISH method, a probe DNA obtained by labeling a cloned gene or DNA fragment with a fluorescent dye or the like is hybridized with a chromosomal DNA on a slide glass, and the molecular hybrid is fluorescent using a fluorescent microscope or the like. In this method, the signal is detected directly on the chromosome. Compared with the conventional autoradiography method, the FISH method is superior in that it does not use a radioactive substance and thus is safe and simple and can obtain a result in a short time. The CISH method is a method for detecting DNA or mRNA on a tissue specimen using a dye such as DAB. Since the fluorescent dye is not used as in the FISH method, it is not necessary to carry out under a fluorescent microscope, and it can be observed under an optical microscope.

As one example of application of the FISH method described above, two or more types of probe DNAs labeled with fluorescent dyes that emit fluorescent colors with different color tones (for example, orange, green, yellow, etc.) are used, and they are simultaneously fluorescent. There is something that maps chromosomes.

In addition, a method for detecting a cancer tissue using such characteristics of the FISH method has been proposed (see, for example, Patent Documents 1 and 2). These detection methods combine the FISH method and other detection methods to perform cancer diagnosis with high accuracy and accurately determine the effectiveness of anticancer drug treatment.

Furthermore, cancer diagnosis methods using the FISH method are being researched and developed. Specifically, it is known that chromosomal abnormalities are observed in cancer cells due to translocation, insertion, inversion, and the like. Such chromosomal abnormalities are considered to be caused by gene disruption and ligation at the DNA level, and the formation of genes that do not normally exist (fusion genes). Therefore, there is a method of diagnosing cancer by identifying a fusion gene peculiar to cancer using the FISH method and detecting the fusion gene.

Here, as a specific example of the FISH method, an outline of the FISH method (indirect method) using biotin as a label is shown in FIG. First, probe DNA is labeled using biotin-dUTP (for example, biotin-1-dUTP manufactured by Roche Applied Science) (S1) to prepare biotin-labeled DNA. Thereafter, the biotin-labeled DNA is heat denatured (S2). At the same time, the chromosome specimen is also heat denatured to prepare single-stranded DNA (S3).

Next, the single-stranded DNA and biotin-labeled DNA are hybridized, and the respective DNAs are complementarily bound (S4). Then, after washing the obtained double strand of biotin-labeled DNA and chromosomal DNA, it is treated with an avidin-FITC (for example, AP-labeled streptavidin manufactured by Roche Applied Science, etc.) solution having high affinity for biotin ( S5). After washing this with a predetermined washing solution, the chromosomal DNA is fluorescently stained, and the locus on the chromosome is observed as a fluorescent signal using a fluorescent microscope. In this way, the molecular hybrid component on the chromosome can be detected directly on the chromosome as a fluorescent signal.

Here, a probe set used in the FISH method when labeling with two kinds of fluorescent dyes will be described. Usually, the target DNA 101 for which the FISH method is used includes a cleavage region 102 including a cleavage point at which the target DNA 101 is cleaved and an important part 103 that is important for pathophysiology, for example, as shown in FIG. ing. The probe set 105 corresponding to the target DNA 101 is composed of two labeled probes 105a and 105b that hybridize with DNA located so as to sandwich the cleavage region 102 of the target DNA 101. The labeled probe 105a is labeled with an orange fluorescent dye, and the labeled probe 105b is labeled with a green fluorescent dye. The length of the target DNA 101 is usually several times (several hundred kb) or more of the labeled probes 105a and 105b.

Using this probe set 105, the FISH method is performed in an environment where the target DNA 101 is present, and the fluorescence signal is observed using a fluorescence microscope. When the target DNA 101 is not cleaved, the target DNA 101 is cleaved. DNAs positioned so as to sandwich the region 102 are positioned in the vicinity of each other. Then, the labeled probes 105a and 105b bound to the DNA located so as to sandwich the cleavage region 102 of the target DNA 101 are also located in the vicinity of each other. As a result, when the target DNA 101 that has not been cleaved is observed with a fluorescence microscope, yellow fluorescence in which orange fluorescence and green fluorescence overlap is observed.

On the other hand, when the target DNA 101 is cleaved by reciprocal translocation or the like and divided into two target DNA pieces, these target DNA pieces are often located at positions separated from each other. In many cases, the labeled probes 105a and 105b bound to the positioned DNA are also located some distance apart. As a result, when the cleaved target DNA is observed with a fluorescence microscope, the orange fluorescence and the green fluorescence hardly overlap, and each fluorescence is observed separately, or When either one goes out of the observation area or one of the target DNA pieces is lost and disappears, only the fluorescence of the labeled probe located in the vicinity of the remaining target DNA piece is observed. It will be.

Therefore, when the target DNA 101 that may be cleaved by reciprocal translocation or the like is observed using the conventional FISH method, basically, orange fluorescence and / or green fluorescence, and yellow Will be observed.

Although the FISH method has been described above as an example, the same applies to the CISH method.

JP 2004-157053 A Special table 2002-542793

However, in the probe set 105 used in the conventional FISH method, as shown in FIG. 20, the DNAs positioned so as to sandwich the cleavage region 102 of the target DNA 101 and the labeled probes 105a and 105b bind to each other. Therefore, since the orange fluorescent dye and the green fluorescent dye are also located at a certain distance, even if the target DNA 101 is not cut, it depends on the relative position and angle between the fluorescent microscope and the target DNA 101. There is a problem that orange and green fluorescence may be observed instead of yellow fluorescence.

As a result, when analyzing fluorescence obtained with a fluorescence microscope, orange fluorescence, green fluorescence, and yellow fluorescence are observed, so that the target DNA 101 that has not been cleaved is mistakenly cleaved. There was a problem that it might end. Regarding this problem, there is a tendency that the possibility of being misidentified increases as the distance between the labeled probe 105a and the labeled probe 105b increases. The same applies to the CISH method.

Further, in the probe set 105 used in the conventional FISH method, the fact that the target DNA 101 is cleaved is determined by the orange fluorescence and the green fluorescence being located apart from each other. Therefore, in the probe set 105, as shown in FIG. 20, is the target DNA 101 cut at the cutting point 107 included in the cutting region 102 or at the cutting point 108 not included in the cutting region 102? There was a problem that could not be judged.

This is a serious problem when, for example, the FISH method is used for diagnosis of cancer. That is, when a cancer is diagnosed by performing the FISH method using such a probe set 105, it is based on pathophysiologically significant cleavage (for example, corresponding to cleavage at the break point 107 included in the cut region 102). Determine whether the fluorescence is the fluorescence of the cleaved target DNA or the fluorescence of the target DNA cleaved by a pathophysiologically insignificant cleavage (for example, corresponding to cleavage at the cleavage point 108 not included in the cleavage region 102). It will not be possible. As a result, as a result of the diagnosis of cancer, there is a problem that although it is actually negative, it is erroneously determined to be positive.

In addition, in the instruction manual of the commercially available probe set, even when the target DNA is not cleaved, orange fluorescence and green fluorescence may be observed with a low probability. In some cases, it is described that it is desirable for each operator to establish the criteria for determining whether or not the item is disconnected.

In view of the circumstances described above, the present invention, when using the FISH method or the CISH method or the like, when observing the color of the fluorescence or coloring regardless of the relative position and angle between the fluorescence microscope and the target DNA. Another object of the present invention is to provide a method for cleaving a target nucleic acid, which can prevent an uncut target DNA from being mistaken for being cleaved.

The inventor of the present invention has conducted extensive research on this problem, and as a result, has found the following innovative method for cleaving and labeling target nucleic acids.

A first aspect of the present invention that solves the above problem is a method for cleaving a nucleic acid sequence to determine whether or not a target nucleic acid sequence having a cleavage region containing at least one cleavage point has been cleaved . labeled with either different identification factor consists of two or more labeled probes, hybridization solution containing a probe set that can be almost entire sequence and hybridization of the target nucleic acid, comprising the steps of contacting the target nucleic acid, the identification A method for cleaving a nucleic acid sequence, comprising the step of causing a factor to function and generating a signal.

In such a first aspect, two or more probe sets labeled with different discriminating agents will hybridize to almost the entire sequence of the target nucleic acid, so that when the target nucleic acid is not cleaved, at least two A signal in which different discrimination factors are mixed (overlapped) (for example, yellow fluorescence when orange and green fluorescent dyes are used in the FISH method) is observed.

On the other hand, when the target nucleic acid is cleaved and divided into target nucleic acid pieces, for example, a mixture of at least two different discrimination factors (for example, two fluorescent dyes of orange and green are used in the FISH method). Yellow fluorescence) and the signal of one of the discriminating factors (for example, when two fluorescent dyes of orange and green are used in the FISH method, orange fluorescence or green fluorescence). Either one of the two colors of fluorescence will be observed, or one of the target nucleic acid pieces will go out of the observation area, or one of the target nucleic acid pieces will be lost and disappear. The signal of the discriminating factor of the remaining target nucleic acid fragment (for example, when two fluorescent dyes of orange and green are used in the FISH method, either orange fluorescence, green fluorescence, or yellow fluorescence) ) It is is will be.

When yellow fluorescence is observed through a filter or the like (including an image processing technique) when the target nucleic acid is not cleaved, orange fluorescence and green fluorescence are observed in the vicinity of the yellow fluorescence.

Therefore, using the first aspect of the present invention, when the target nucleic acid is not cleaved, a signal mixed with a discrimination factor is observed, and when the target nucleic acid is cleaved, the conventional FISH method is Differently, either a signal mixed with a discriminating factor and a signal of one of the discriminating factors are observed, or a signal of either one of the discriminating factors or a signal mixed with the discriminating factors is observed. It is possible to prevent a target nucleic acid that has not been cleaved from being mistaken for being cleaved.

In addition, since the probe set hybridizes to almost the entire sequence of the target nucleic acid, the amount (color, intensity, etc.) of the target nucleic acid discrimination factor signal that is not cleaved and the target nucleic acid discrimination factor that is cleaved The cleavage position of the target nucleic acid can be estimated by comparing the amount of the signal.

According to a second aspect of the present invention, there is provided the method for cleaving and labeling a nucleic acid sequence according to the first aspect, wherein at least one of the labeled probes is hybridized with a cleavage region of the nucleic acid sequence.

In the second aspect, in addition to the effects obtained in the first aspect, it can be determined more clearly whether the nucleic acid sequence is cleaved at the cleavage region or at other sites.

According to a fourth aspect of the present invention, the nucleic acid sequence further comprises at least one important part, a labeled probe that hybridizes to the cleavage region of the nucleic acid sequence, and a nucleic acid sequence positioned so as to sandwich the important part with respect to the cleavage region The method for cleaving and labeling a nucleic acid sequence according to any one of the first to third aspects, wherein the labeled probe that hybridizes to the portion is a different labeled probe.

In the fourth aspect, it can be determined whether the nucleic acid sequence is cleaved at a breakpoint that is not an important part or is cleaved elsewhere.

The fifth aspect of the present invention is the method for cleaving and labeling a nucleic acid sequence according to any one of the first to fourth aspects, characterized in that the probe set has a mixing part labeled in a state where different discrimination factors are mixed. It is.

In the fifth aspect, when the target nucleic acid is cleaved and divided into target nucleic acid pieces, hybridization with the probe set causes at least one target nucleic acid piece to contain either signal of the labeling factor. At the same time, by passing through a filter or the like, a signal in which discrimination factors are mixed (overlapped) from the mixing part of the probe set is also observed. On the other hand, although there is a possibility of hybridization with a nucleic acid other than the target nucleic acid depending on the labeled probe, only the signal of the labeling factor bound to the labeled probe is observed from the nucleic acid.

Therefore, by using the fifth aspect of the present invention, even when the labeled probe is hybridized with a nucleic acid other than the target nucleic acid, by confirming the signal mixed with the identification factor from the mixing part, Since it can be determined whether the target nucleic acid is cleaved or a nucleic acid other than the target nucleic acid, the target nucleic acid that is not cleaved is misidentified as cleaved. Can be more reliably prevented.

A sixth aspect of the present invention is the method for cleaving and labeling a nucleic acid sequence according to the fifth aspect , wherein the mixing part is hybridized with a part or all of the important part.

In the sixth aspect, it can be more reliably determined whether the nucleic acid sequence is cleaved at the breakpoint included in the cleavage region or is cleaved elsewhere.

A seventh aspect of the present invention is the method according to any one of the first to fourth aspects, wherein one or a plurality of labeled probes labeled with the same discriminating factor hybridize with almost the entire sequence of the target nucleic acid. A method for cleaving and labeling nucleic acid sequences.

In the seventh aspect, since the target nucleic acid is cleaved at a portion corresponding to the portion labeled with the same discriminating factor, the amount of the signal of the discriminating factor of the target nucleic acid that has not been cleaved is determined. By comparing the amount of signal of the target nucleic acid discriminating factor, the cleavage position of the target nucleic acid can be estimated more accurately.

In addition, since the target nucleic acid is not cleaved at the part corresponding to the boundary part of each discrimination factor, when the target nucleic acid is not cleaved, a signal mixed with each discrimination factor is observed, and the target nucleic acid is cleaved. In this case, either a signal of the discriminating factor and a signal mixed with each discriminating factor are observed, or one of the signals is observed. It can prevent more reliably that it is mistaken as what is cut | disconnected.

The eighth aspect of the present invention is a step of detecting a signal, wherein different discrimination factors are pigments of different colors, and the step of causing the discrimination factor to function and generating a signal is a step of irradiating the pigment with light to develop a color. The method for cleaving and labeling a nucleic acid sequence according to any one of the first to seventh aspects, wherein the method is a step of detecting color development.

In the eighth aspect, the method for cleaving and labeling a nucleic acid sequence can be performed by the same operation as the conventional CISH method.

A ninth aspect of the present invention is a fluorescent dye in which different discriminating factors emit different fluorescent colors, the step of functioning the discriminating factor to emit a signal is the step of causing the fluorescent pigment to fluoresce, and the step of detecting the signal is fluorescent. 8. The method for cleaving and labeling a nucleic acid sequence according to any one of claims 1 to 7 , wherein the method is a step of detecting.

In the ninth aspect, a method for cleaving and labeling a nucleic acid sequence can be performed by the same operation as in the conventional FISH method.

A tenth aspect of the present invention consists of a dye that develops two different hues in the modified Munsell modified color system or a fluorescent dye that emits two different hues of fluorescent colors in the modified Munsell modified color system, Among the different hues, the cut point is a straight line connecting the hue and the central point of the modified Munsell hue ring, and a straight line connecting the hue of the mixed color when the two different hues are mixed and the central point of the modified Munsell hue ring. 10. The method for cleaving and labeling a nucleic acid sequence according to claim 8 or 9 , wherein the nucleic acid sequence corresponds to a portion labeled with a dye having a larger hue formed by or a fluorescent dye.

In the tenth aspect, a straight line connecting the hue and the center point of the corrected Munsell hue ring and a straight line connecting the hue of the mixed color when two different hues are mixed and the central point of the corrected Munsell hue ring are formed. Dye or fluorescent dye having a hue with a larger angle (hue angle) can more easily discriminate the difference from the mixed color than a dye or fluorescent dye having a smaller hue angle. . As a result, when the target nucleic acid is not cleaved, a signal mixed with each discrimination factor is observed, and when the target nucleic acid is cleaved, one of the discrimination factors and a signal mixed with each discrimination factor are observed. Since it will be observed, it can prevent more reliably that the target nucleic acid which is not cut | disconnected is misidentified as having been cut | disconnected.

In the present invention, “nucleic acid” refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA) and others having the same functions as these, and the sequence and length thereof are particularly limited. Instead, it may be one that actually exists in nature, one that has been generated by mutation (for example, a fusion gene, etc.), or one that has been artificially produced. An example of a method for artificially synthesizing a nucleic acid is a solid phase synthesis method.

In the present invention, the “cleavage region” means a part of a nucleic acid sequence that is assumed to include a breakpoint by a person who carries out the present invention, but when the breakpoint is unknown, the whole target nucleic acid is meant. .

Furthermore, in the present invention, the “important part” refers to a part of a nucleic acid sequence that is determined to be important by a person who performs the present invention. For example, when the present invention is applied to a method for diagnosing cancer, pathophysiologically. It refers to a part of an important nucleic acid sequence (for example, a kinase domain). In the present invention, at least one important part of the nucleic acid sequence is sufficient, and two or more important parts may be present in the nucleic acid sequence. In the case where there are a plurality of important parts in the nucleic acid sequence, the present invention may be carried out with a person who implements the present invention more important as an important part of the present invention.

3 is a conceptual diagram of a probe set according to Embodiment 1. FIG. It is a conceptual diagram in case the target nucleic acid is cut | disconnected. It is a conceptual diagram at the time of hybridizing the probe set which concerns on Embodiment 1 when the target nucleic acid is cut | disconnected. 2 is a conceptual diagram of a probe set according to Embodiment 1. FIG. 2 is a conceptual diagram of a probe set according to Embodiment 1. FIG. It is a conceptual diagram at the time of hybridizing the probe set which concerns on Embodiment 1 when the target nucleic acid is cut | disconnected. 2 is a conceptual diagram of a probe set according to Embodiment 1. FIG. 6 is a conceptual diagram of a labeled probe according to Embodiment 2. FIG. 6 is a conceptual diagram of a probe set according to Embodiment 2. FIG. It is a conceptual diagram at the time of hybridizing the probe set which concerns on Embodiment 2 when the target nucleic acid is cut | disconnected. FIG. 6 is a conceptual diagram of a probe set according to a third embodiment. FIG. 6 is a conceptual diagram of a probe set according to a third embodiment. It is a conceptual diagram of the probe set which concerns on Embodiment 4. It is a conceptual diagram of the probe set which concerns on Embodiment 4. It is a conceptual diagram of the probe set which concerns on Embodiment 4. It is a conceptual diagram of the probe set which concerns on other embodiment. It is a fluorescence image figure of an Example. It is a fluorescence image figure of a comparative example. It is a schematic explanatory drawing of the conventional FISH method. It is a conceptual diagram of the conventional probe set.

The method for cleaving and labeling a nucleic acid sequence according to the present invention comprises a hybridization solution comprising a probe set comprising two or more labeled probes labeled with different identification factors and capable of hybridizing to almost the entire sequence of the target nucleic acid, and the target The process includes two steps: a step of bringing a nucleic acid into contact (first step) and a step of causing the discrimination factor to function and outputting a signal (second step). Hereinafter, each process is explained in full detail. In addition, about these processes, except the structure of a probe set, it can carry out similarly to the conventional FISH method and CISH method.

First, the first step will be described. The hybridization solution used in the first step is composed of two or more labeled probes labeled with different identification factors, and includes a probe set that can hybridize to almost the entire sequence of the target nucleic acid. There is no particular limitation.

Here, as an identification factor, for example, a fluorescent dye used in the FISH method or a dye used in the CISH method, light (including ultraviolet rays and infrared rays) is emitted and transmitted by some other stimulus. There is no particular limitation as long as it can be (including by absorbing some wavelengths).

The labeled probe is not particularly limited as long as it can hybridize to the target nucleic acid and is labeled with a labeling factor. For example, deoxyribonucleic acid (DNA) labeled with a labeling factor, ribonucleic acid (RNA), peptide nucleic acid (PNA), etc., as long as they have the same function as these, the sequence and length thereof are particularly limited. Instead, it may exist in nature or may be artificially produced.

It should be noted that DNA labeled with a labeling factor is DNA or the like in which a discrimination factor is directly bound (directly bound), but the discrimination factor is a protein, antibody, etc. It may be a DNA or the like that is indirectly bound via a chain (including those that are superimposed).

A probe set that can hybridize to almost the entire sequence of the target nucleic acid is not only a probe set that can completely hybridize to the entire sequence of the target nucleic acid, but also a portion that cannot hybridize to the entire sequence of the target nucleic acid. Including the probe set described below.

Therefore, a hybridization solution containing such a probe set contains other components such as components contained in commercially available hybridization reagents used in, for example, the FISH method and the CISH method. May be.

Hereinafter, the probe set used in the present invention will be described taking the FISH method using two types of fluorescent dyes as an example. The CISH method can be considered similarly.
(Embodiment 1)

FIG. 1 shows a schematic diagram of a probe set 5 when hybridized with a target nucleic acid 1 having a cleavage region 2 containing at least one cleavage point 7 and an important part 3. As shown in this figure, the probe set 5 used in the present invention is composed of labeled probes 5a and 5b. Unlike the probe set used in the conventional FISH method, the entire sequence of the target nucleic acid 1 is shown. Can be hybridized with. The labeled probe 5a is labeled with an orange fluorescent dye, and the labeled probe 5b is labeled with a green fluorescent dye.

Therefore, when the target nucleic acid 1 and the probe set 5 are hybridized and then the fluorescent dye is fluorescent by performing the second step described later, the labeled probes 5a and 5b labeled with the respective fluorescent dyes are located in the vicinity. Therefore, the orange fluorescence of the labeled probe 5a and the green fluorescence of the labeled probe 5b overlap, and yellow fluorescence is observed. However, orange or green fluorescence may be observed in the vicinity of the yellow fluorescence.

Here, the probe set 5 is designed so that it can hybridize with the target nucleic acid 1. For example, as shown in FIG. 1, the probe set 5 is composed of two labeled probes 5a and 5b that do not overlap with the target nucleic acid 1 when hybridized with the target nucleic acid 1. Designed.

The complementarity between the labeled probes 5a and 5b and the corresponding sequence of the target nucleic acid 1 is preferably 100%, but is not limited to this as long as it retains the function of specifically binding to the target nucleic acid 1. Depending on the aspect of the target nucleic acid 1, the complementarity may be less than 100%, for example 90%. Moreover, it is preferable that the glycine (G) / cytosine (C) contents of the labeled probes 5a and 5b are equal so that the labeled probes 5a and 5b have similar thermal stability. These labeled probes 5a and 5b may be combinations of commercially available products, newly prepared products, artificially prepared by a solid phase synthesis method or the like, or combinations thereof. Also good.

Next, the second step will be described. This 2nd process will not be specifically limited if a discrimination factor is made to function and a signal can be output. For example, when the discriminating factor is a fluorescent dye, the fluorescent dyes of the labeled probes 5a and 5b may be made to fluoresce using a laser device that can emit light having a wavelength that allows each fluorescent dye to fluoresce. If the identification factor is a dye, the dye of the probe set may be colored using an apparatus that can emit visible light.

The signal obtained from the identification factor through a fluorescence microscope or the like may be observed visually or may be observed using an observation device. The observation instrument is not particularly limited as long as it can detect the signal obtained in the second step. For example, when the discriminating factor is a fluorescent dye, fluorescence is obtained using a commercially available fluorescence microscope apparatus used in the conventional FISH method, that is, an optical apparatus, digital imaging hardware, computer hardware, and computer software. It can be observed. When the discriminating factor is a pigment, the color is observed using a commercially available optical microscope apparatus used in the conventional CISH method, that is, an optical apparatus, digital imaging hardware, computer hardware, and computer software. can do.

Here, the signals observed when the target nucleic acid 1 is not cleaved and when it is cleaved will be described using the FISH method described in the first step as an example.

As described above, when the target nucleic acid 1 is not cleaved, yellow fluorescence in which orange fluorescence and green fluorescence are mixed is observed.

On the other hand, for example, when the target nucleic acid 1 is cleaved at the break point 7 included in the cleavage region 2 shown in FIG. 1 due to the influence of reciprocal translocation, the target nucleic acid pieces 1A and 1B are divided as shown in FIG. Is done. Then, when the first step is performed in this state, as shown in FIG. 3, each of the target nucleic acid pieces 1A and 1B and probe set pieces 5A and 5B obtained by cutting the probe set 5 at the cutting point 7 are obtained. Hybridization will occur. The probe set piece 5A is labeled with an orange fluorescent dye and a green fluorescent dye, and the probe set piece 5B is labeled only with a green fluorescent dye. These target nucleic acid pieces 1A and 1B are often located away from each other.

As a result, when the target nucleic acid 1 is thus cleaved and divided into the target nucleic acid pieces 1A and 1B, the orange fluorescence (and a small amount of green fluorescence partially overlapped with the yellow color of the probe set piece 5A) ) And the green fluorescence of the probe set piece 5B, or one of the target nucleic acid pieces 1A and 1B goes out of the observation region, or one of the target nucleic acid pieces 1A and 1B. As a result of loss and disappearance, only one fluorescence is observed.

Therefore, unlike the conventional FISH method, only the yellow fluorescence of the probe set 5 is observed when the target nucleic acid 1 is not cleaved, and the orange of the probe set piece 5A when the target nucleic acid 1 is cleaved. Of the fluorescence (and yellow fluorescence with a small amount of green fluorescence partially overlapped) and the green fluorescence of the probe set piece 5B, or only one of them is observed. It is possible to prevent the target nucleic acid 1 that has not been cleaved from being mistaken for being cleaved.

In addition, since the probe set 5 can hybridize with the entire sequence of the target nucleic acid 1, the fluorescence color and intensity of the target nucleic acid 1 that has not been cleaved and the fluorescence of the cleaved target nucleic acid fragments 1A and 1B. By comparing the color and its intensity, the break point 7 of the target nucleic acid 1 can be estimated.

The effect described above is that, like a probe set 5 as shown in FIG. 1, the probe set has a labeled probe 5 b that hybridizes to the cleavage region 2 of the target nucleic acid 1, and an important part 3 with respect to the cleavage region 2. Not only the case where the labeled probe 5a that hybridizes to the portion of the target nucleic acid 1 positioned so as to be sandwiched is composed of different labeled probes, but also the cleavage region of the target nucleic acid 1 as in the probe set 5 'as shown in FIG. The labeled probe 5b ′ that hybridizes to 2 and the labeled probe 5b ′ that hybridizes to the portion of the target nucleic acid 1 positioned so as to sandwich the important part 3 with respect to the cleavage region 2 are the same labeled probe (from the labeled probe 5b). It is clear that the same phenomenon occurs even in the case of a long-term probe).

Further, in this example, as described above, the probe set 5 ′ composed of the labeled probe 5b ′ and the labeled probe 5a ′ that hybridizes to the cleavage region 2 of the target nucleic acid 1, and the target nucleic acid 1 cleaved at the cleavage point 7 And are hybridized. Therefore, when the fluorescent dye is subsequently made fluorescent, orange fluorescence (and yellow fluorescence with a small amount of green fluorescence partially overlapped) and green fluorescence are observed (see FIG. 4).

On the other hand, if the target nucleic acid 1 is cleaved at a cutting point 8 that is not included in the cleavage region 2 and is not normally cleaved as shown in FIG. 5 for some reason, as shown in FIG. It is divided into pieces 1A ′ and 1B ′. Then, when the first step is performed in this state, the respective target nucleic acid pieces 1A ′ and 1B ′ and the probe set pieces 5A ′ and 5B ′ obtained by cutting the probe set 5 ′ at the cutting point 8 are hybridized. Will do. Then, when the fluorescent dye is made to fluoresce in this state, the orange fluorescence of the probe set piece 5A ′ and the green fluorescence of the probe set piece 5B ′ (the yellow fluorescence in which the orange fluorescence partially overlaps the green fluorescence) Fluorescence) is observed.

Therefore, by observing the fluorescence in the present embodiment, whether the target nucleic acid 1 is cleaved at the cutting point 7 included in the cutting region 2 or is cut at the cutting point 8 not included in the cutting region 2 Can be judged.

Further, as shown in FIG. 7, the labeled probe 15b that hybridizes to the cleavage region 2 and the important part 3 and the important part 3 so that the arrangement of the labeled probes is opposite to that of the probe set 5 shown in FIG. Alternatively, the probe set 15 may be composed of a labeled probe 15a that hybridizes to a portion of the target nucleic acid 1 positioned so as to sandwich the cleavage region 2 therebetween.

In the case where the probe set 15 is configured in this way, when the target nucleic acid 1 is cleaved at the breakpoint 7, the orange fluorescence (and partly) of the labeled probe hybridized with the portion not including the important portion 3 is included. Yellow fluorescence in which a small amount of green fluorescence overlaps) and green fluorescence of the labeled probe hybridized with the portion containing the important portion 3 is observed.

Therefore, when the probe set 15 is configured in this way, by observing a green single-color fluorescence, it is meaningful in terms of pathophysiology (for example, when the present invention is applied to a method for diagnosing cancer). It can be determined that a certain disconnection has occurred.
(Embodiment 2)

In Embodiment 1, the probe set 5 is designed to be composed of two labeled probes 5a and 5b that do not overlap with the target nucleic acid 1 as shown in FIG. Thus, it may be designed to be composed of two labeled probes 25a and 25b so that an overlapping mixed portion α is formed when hybridized with the target nucleic acid 1. As shown in FIG. 9, the mixing part α of the probe set 25 is usually labeled in a state where these fluorescent dyes are mixed.

Then, when the first step described above is performed when the target nucleic acid 1 is cleaved using the probe set 25, the target nucleic acid pieces 1A and 1B and the probe set 25 are connected as shown in FIG. The probe set pieces 25A and 25B that have been cut at the cutting point 7 are hybridized respectively.

Then, in the case where the target nucleic acid 1 is cleaved and divided into the target nucleic acid pieces 1A and 1B, the orange color or the yellow fluorescence and the green fluorescence of the probe set piece 25A overlap with each other as a whole. Yellow fluorescence with green fluorescence overlapped on the part and the green fluorescence of the probe set piece 25B are observed, or one of the target nucleic acid pieces 1A, 1B goes out of the observation region, or the target nucleic acid When either one of the pieces 1A and 1B is lost and disappears, only one of the fluorescences is observed.

In the second embodiment, since the probe set 25 is configured such that the mixing portion α is formed at a location corresponding to the important portion 3, the yellow fluorescence corresponding to the mixing portion α is adjacent to the orange fluorescence. Is preferable because it can be more reliably determined whether the target nucleic acid 1 is cleaved at the breakpoint 7 or at any other place. However, the probe set according to the second embodiment is not limited to this configuration.

Further, when the probe set piece 25A is observed in more detail using a filter or an image processing technique, the yellow fluorescence of the mixing portion α can be observed together with the orange fluorescence and the green fluorescence. Therefore, by confirming them, it can be more reliably determined whether the target nucleic acid 1 is cleaved at the breakpoint 7 included in the cleavage region 2 or is cleaved elsewhere.
(Embodiment 3)

The probe set 5 of Embodiment 1 is configured with two labeled probes 5a and 5b whose sequence length matches the entire sequence of the target nucleic acid 1, but the probe set according to the present invention is not limited thereto. For example, as shown in FIG. 11, the probe set 35 may be composed of a labeled probe 35a and a labeled probe 5b having a nucleic acid sequence longer than that of the labeled probe 5a of the first embodiment. Even with such a probe set 35, the same effects as those of the probe set 5 of Embodiment 1 can be obtained.

In addition, as shown in FIG. 12, the labeled probe 45b that can hybridize with almost the entire sequence of the target nucleic acid 1, and a portion that hybridizes with the remaining portion 46 of the target nucleic acid 1, and the labeled probe 5a of the first embodiment. The probe set 45 may be configured with a labeled probe 45a having a longer nucleic acid sequence. The labeled probe 45b may be composed of a nucleic acid sequence longer than the target nucleic acid 1.

Even if the probe set 45 is configured in this manner, the same effect as in the case of the probe set 5 of Embodiment 1 can be obtained, and the labeled probe 45b has the same length as the target nucleic acid 1; Measure the intensity of fluorescence of the green fluorescent dye labeled on the green fluorescent dye of the target nucleic acid 1 when not cleaved, and the intensity of the green fluorescent dye of the target nucleic acid 1 when cleaved By comparing the intensity of fluorescence, the cleavage position of the target nucleic acid 1 can be estimated more accurately than in the case of the probe set 5 of Embodiment 1.
(Embodiment 4)

Unlike the probe set 5 that can hybridize with the entire nucleic acid sequence of Embodiment 1, as shown in FIG. 13, the labeled probe 5a and the labeled probe are formed so that a region 56 that cannot be hybridized is formed in a portion corresponding to the cleavage region 2. The probe set 55 may be configured with a labeled probe 55b having a sequence shorter than 5b.

In such a probe set 55, when the target nucleic acid 1 is hybridized, a space is formed between the labeled probe 5a and the labeled probe 55b, and a region 56 that cannot be hybridized with the target nucleic acid 1 is formed. Since it can hybridize with almost the entire sequence of the nucleic acid 1, the same effect as in the case of the probe set 5 of Embodiment 1 can be obtained.

The length of the region 56 that cannot be hybridized is not limited as long as the effects of the present invention can be obtained, but is preferably 100 kb or less, more preferably 50 kb or less, and even more preferably as it becomes 40 kb or less, 30 kb or less, or 20 kb or less. And 10 kb or less is most preferable. The effect of the present invention becomes more prominent as the region 56 that cannot be hybridized becomes shorter.

Further, when examining whether or not the target nucleic acid 1 whose breakpoint 7 is known is cleaved, as shown in FIG. 13, a region 56 that cannot hybridize with the target nucleic acid 1 is formed in the vicinity of the breakpoint 7. In addition, the probe set 55 may be constituted by the labeled probe 5a and the labeled probe 55b.

By configuring the probe set 55 in this manner, when the target nucleic acid 1 is not cleaved, yellow fluorescence in which orange fluorescence and green fluorescence are mixed is observed. Since the orange fluorescence and the green fluorescence are separately observed, it is possible to more reliably prevent the target nucleic acid 1 that has not been cleaved from being mistaken for being cleaved.

In this example, the probe set 55 is configured as described above in order to create a space between the labeled probe 5a and the labeled probe 55b. However, as shown in FIG. The probe set 65 is composed of a labeled probe 65a and a labeled probe 5b having a shorter sequence than the labeled probe 5a so that a region 66 that cannot hybridize with the target nucleic acid 1 is formed on the end side of the target nucleic acid 1 when hybridized. May be. Even if the probe set 65 is configured in this manner, the same effect as that of the probe set 5 of the first embodiment can be obtained.

Furthermore, as shown in FIG. 15, the probe set 75 may be configured with a labeled probe 75b and a labeled probe 5a in which a part 76 of the labeled probe 5b of Embodiment 1 is missing. When the labeled probe 5b has a portion capable of hybridizing with a nucleic acid other than the target nucleic acid 1, a probe set 75 is constituted by the labeled probe 75b and the labeled probe 5a excluding the portion, whereby a nucleic acid other than the target nucleic acid 1 is formed. Since fluorescence from the sequence can be prevented, it is possible to more reliably prevent the target nucleic acid 1 that has not been cleaved from being mistaken for being cleaved.
(Other embodiments)

In the probe set described above, the probe set is configured by two labeled probes. However, the configuration of the probe set according to the present invention is not limited to this, and more labeled probes, for example, as shown in FIG. Three labeled probes 85a, 85b, and 85c may be used. Even if the probe set 85 is configured in this way, the same effect as that of the probe set 5 of the first embodiment can be obtained.

In this case, the fluorescent dyes bound to the respective labeled probes 85a, 85b, 85c may be fluorescent dyes that emit different fluorescence for each of the labeled probes 85a, 85b, 85c. For example, the fluorescent dyes of the labeled probes 85b The dye may be the same as the fluorescent dye of either the labeled probe 85a or the labeled probe 85c (two color fluorescent dyes in the entire probe set 85). When the probe set 85 labeled with a fluorescent dye that emits different fluorescence for each labeled probe 85a, 85b, 85c is used, the positional relationship of the target nucleic acid 1 that hybridizes with each labeled probe 85a, 85b, 85c becomes clearer. The cleavage position of the target nucleic acid 1 can be estimated more accurately than in the case of the probe set 5 of the first embodiment.

Although other embodiments have been described above, the probe set according to the present invention is not limited to this, and includes a probe set configured by combining them.

Also, the identification factor develops two different hues (for example, orange and green) in the modified Munsell modified color system, or the fluorescent dye has two different hues in the modified Munsell modified color system (for example, orange). When the target nucleic acid breaks, the target nucleic acid cleavage point is a mixture of these different hues and the straight line connecting the hue and the central point of the modified Munsell hue ring. Colorant (for example, yellow if the different hues are orange and green) and the dye of the hue with the larger angle (hue angle) formed by the straight line connecting the center point of the modified Munsell hue ring (hue angle) For example, it is preferred to design the probe set so that it is in the nucleic acid sequence corresponding to the portion labeled with green) or a fluorescent dye (eg, green).

The hue dye having a larger hue angle (for example, green) or the fluorescent dye (for example, green) is the one having the hue hue having the smaller hue angle (for example, orange) or the fluorescent dye (for example, orange). The difference from the color mixture can be more easily identified than the method. As a result, when the target nucleic acid is not cleaved, a signal mixed with each discrimination factor is observed, and when the target nucleic acid is cleaved, one of the discrimination factors and a signal mixed with each discrimination factor are observed. Since it will be observed, it can prevent more reliably that the target nucleic acid which is not cut | disconnected is misidentified as having been cut | disconnected.

In order to detect the rearrangement of the ALK gene, hybridization was performed using RP11-984I21, RP11-62B19 and RP11-701P18 labeled with FITC as labeled probes, and then the conventional FISH method A fluorescent image was created by performing the same operation as described above. RP11-984I21, RP11-62B19 and RP11-701P18 are identification numbers in the human male RP-11 BAC library.

FIG. 17 shows a fluorescence image diagram of the example (negative example). As can be seen from this figure, for all fluorescence, orange fluorescence and green fluorescence overlap and yellow fluorescence (orange or green fluorescence may be observed in the vicinity of yellow fluorescence). I can see it. Here, negative refers to a state in which no mutual translocation or the like has occurred and the target nucleic acid has not been cleaved, and positive refers to a state in which a mutual translocation or the like has occurred and the target nucleic acid has been cleaved. The results were the same when using a labeled probe labeled with FITC and TexRed reversed.

Comparative example

On the other hand, in order to detect the rearrangement of the ALK gene, hybridization was performed using LSI ALK Dual Color as an identification probe, and then the same operation as in the conventional FISH method was performed in the same manner as in Example to obtain a fluorescence image. Created.

FIG. 18 shows a fluorescence image diagram (negative example) of the comparative example. As can be seen from this figure, it can be seen that some of the fluorescence is visible in orange and green. Therefore, if the target nucleic acid is cleaved, it may be misjudged as positive. This is because in the conventional probe set, the orange fluorescent dye and the green fluorescent dye are located at some distance from each other, so that even if the target nucleic acid is not cleaved, the fluorescence microscope and the target nucleic acid It is considered that orange fluorescence and green fluorescence appear by chance depending on the relative position and angle.

As described above, by using the present invention, it is possible to prevent a target nucleic acid that has not been cleaved from being mistaken for being cleaved.

The present invention is used for diagnosis methods (including cancer diagnosis methods) in which the conventional FISH method or the like has been used, DNA or RNA identification methods / specific methods, nucleic acid cleavage position estimation methods, and the like. Can do.

1,101 target nucleic acid 1A, 1B, 1A ', 1B' target nucleic acid piece 2, 102 cleavage region 3, 103 important part 7, 8, 107, 108 cleavage point 5, 15, 25, 35, 45, 55, 65, 75, 85, 105 Probe sets 5A, 5B, 5A ′, 5B ′, 25A, 25B Probe set pieces 5a, 5b, 5a ′, 5b ′, 15a, 15b, 25a, 25b, 35a, 45a, 45b, 55b, 65a , 75b, 85a, 85b, 85c, 105a, 105b Labeled probe α mixing section

Claims (17)

Consisting of two or more different sequences of labeled probes labeled with one of two different discriminators,
At least one labeled probe is labeled with a different identifier than the other labeled probes;
Using a probe set in which the labeled probes of the two or more different sequences can hybridize to almost the entire sequence of the target nucleic acid,
A cutting method for labeling a nucleic acid sequence, wherein the target nucleic acid sequence to determine whether it is cut with at least one cutting region including a cutting point and at least one key portion,
The probe set includes a mixing part labeled in a state in which different identification factors are mixed when hybridized with the nucleic acid sequence,
Contacting the hybridization solution containing the probe set with the target nucleic acid;
Causing the discriminating factor to function and outputting a signal;
And a step of determining whether or not the pathophysiologically significant cleavage is based on the signal . 2. The method for cleaving and labeling a nucleic acid sequence according to claim 1, wherein the mixing part hybridizes with at least part of the important part side of the cleavage region. Consists of two or more different sequences of labeled probes labeled with either of two different discriminating factors, wherein at least one labeled probe is labeled with a different discriminating factor from the other labeled probes, said two or more different Using a probe set that allows the labeled probe of the sequence to hybridize to almost the entire sequence of the target nucleic acid,
A cutting method for labeling a nucleic acid sequence, wherein the target nucleic acid sequence to determine whether it is cut with at least one cutting region including a cutting point and at least one key portion,
And one and / or hybridization solution other identification factor-labeled probe comprising at least a portion capable of hybridizing said probe set of the cutting area, the step of contacting, with the target nucleic acid,
Causing the discriminating factor to function and outputting a signal;
And a step of determining whether or not the pathophysiologically significant cleavage is based on the signal . The labeled probe identified by one of the identification factors is hybridized with the whole of the cleavage region and the part of the nucleic acid sequence including at least a part of the region sandwiched between the cleavage region and the important part. A method for cleaving and labeling a nucleic acid sequence according to claim 3. The labeled probe identified by one of the identification factors includes all or a part of the cleavage region, but does not include the important part, and the part of the nucleic acid sequence that does not contain the region between the cleavage region and the important part. Hybridize,
The labeled probe identified by the other identification factor hybridizes with the portion of the nucleic acid sequence including at least the important portion and the region sandwiched between the cleavage region and the important portion. A method for cleaving and labeling the described nucleic acid sequence. A probe labeled with the one and / or the other discrimination factor can hybridize with a part of the cleavage region,
The probe set includes a portion that cannot hybridize with the target nucleic acid;
The portion that cannot hybridize with the target nucleic acid is in the cleavage region between the labeled probe identified by the one identification factor and the labeled probe identified by the other identification factor, or at the cleavage point. In the vicinity,
And the length is 10KB or less
The method for cleaving and labeling a nucleic acid sequence according to any one of claims 3 and 5. Consisting of three or more different sequences of labeled probes labeled with any of the three different discriminators, at least three of the labeled probes being labeled with any of the three different discriminators, Using a probe set in which labeled probes of two or more different sequences can hybridize to almost the entire sequence of the target nucleic acid,
A cutting method for labeling a nucleic acid sequence, wherein the target nucleic acid sequence to determine whether it is cut with at least one cutting region including a cutting point and at least one key portion,
A probe labeled with a first discriminating factor can hybridize with the entire cleavage region and a part of the important part;
A probe labeled with a second discriminant can at least hybridize with the remainder of the critical portion;
A hybridization solution comprising the probe set capable of hybridizing a probe labeled with a third discriminating factor to a part of the target nucleic acid located on the opposite side of the cleavage region, and the target nucleic acid; A step of contacting
Causing the discriminating factor to function and outputting a signal;
Determining whether a pathophysiologically meaningful disconnection based on the signal; and
A method for cleaving and labeling a nucleic acid sequence, comprising: The different discriminating factors are pigments of different colors;
The step of causing the discrimination factor to function and generating a signal is a step of irradiating the dye with light to develop a color,
The method for cleaving and labeling a nucleic acid sequence according to any one of claims 1 to 7 , wherein the step of detecting the signal is a step of detecting the color development. The different discrimination factors are fluorescent dyes that emit different fluorescent colors;
The step of causing the discrimination factor to function and outputting a signal is a step of causing the fluorescent dye to fluoresce,
The method for cleaving and labeling a nucleic acid sequence according to any one of claims 1 to 7 , wherein the step of detecting the signal is a step of detecting the fluorescence. The dye comprises two different hues in the modified Munsell modified color system or the fluorescent dye emits two different hues of fluorescent colors in the modified Munsell modified color system;
Among the different hues, the cut point is a straight line connecting the hue and the central point of the modified Munsell hue ring, and the hue of the mixed color and the central point of the modified Munsell hue ring when the two different hues are mixed. 10. The method for cleaving and labeling a nucleic acid sequence according to claim 8 or 9 , wherein the nucleic acid sequence corresponds to a portion labeled with a dye having a larger hue formed by a straight line connecting to the dye or a fluorescent dye. . Consisting of two or more different sequences of labeled probes labeled with one of two different discriminators,
At least one labeled probe is labeled with a different identifier than the other labeled probes;
The two or more different sequence labeled probes can hybridize to almost the entire sequence of a target nucleic acid having a cleavage region containing at least one cleavage point and at least one important portion, and the target nucleic acid is cleaved. A probe set for determining pathophysiological cleavage that can determine whether or not the cleavage is pathophysiologically meaningful,
A probe set for determining pathophysiological cleavage, comprising a mixing part labeled in a state in which different discrimination factors are mixed when hybridized with the nucleic acid sequence. The probe set for pathophysiological cleavage determination according to claim 11, wherein the mixing part hybridizes with at least a part of the important part side of the cleavage region. Consisting of two or more different sequences of labeled probes labeled with one of two different discriminators,
At least one labeled probe is labeled with a different identifier than the other labeled probes;
When the labeled probes of the two or more different sequences can hybridize with almost the entire sequence of the target nucleic acid having a cleavage region containing at least one cleavage point and at least one important part, and the target nucleic acid has been cleaved Further, a probe set for determining pathophysiological cleavage that can determine whether or not the cleavage is pathophysiologically meaningful,
A probe set for determining pathophysiological cleavage, wherein a probe labeled with one and / or the other discrimination factor can hybridize with at least a part of the cleavage region. The labeled probe identified by one of the identification factors is capable of hybridizing with the whole of the cleavage region and the portion of the nucleic acid sequence including at least a part of the region sandwiched between the cleavage region and the important part. The probe set for pathophysiological cutting determination according to claim 13. The labeled probe identified by one of the identification factors includes all or a part of the cleavage region, but does not include the important part, and the part of the nucleic acid sequence that does not contain the region between the cleavage region and the important part. Can hybridize,
The labeled probe identified by the other identification factor can hybridize with the nucleic acid sequence portion including at least the important portion and the region sandwiched between the cleavage region and the important portion. Probe set for judgment of pathophysiological cleavage of A probe labeled with the one and / or the other discrimination factor can hybridize with a part of the cleavage region,
The probe set includes a portion that cannot hybridize with the target nucleic acid;
The portion that cannot hybridize with the target nucleic acid is in the cleavage region between the labeled probe identified by the one identification factor and the labeled probe identified by the other identification factor, or at the cleavage point. In the vicinity,
And the length is 10KB or less
The probe set for pathophysiological cutting determination according to any one of claims 13 and 15, wherein: Consisting of three or more different sequences of labeled probes labeled with one of three different discriminators,
At least three of the labeled probes are labeled with any of the three different identifiers;
When the labeled probes of the three or more different sequences can hybridize with almost the entire sequence of the target nucleic acid having a cleavage region containing at least one cleavage point and at least one important part, and the target nucleic acid has been cleaved Further, a probe set for determining pathophysiological cleavage that can determine whether or not the cleavage is pathophysiologically meaningful,
A probe labeled with a first discriminating factor can hybridize with the entire cleavage region and a part of the important part;
A probe labeled with a second discriminant can at least hybridize with the remainder of the critical portion;
A probe set for determining pathophysiological cleavage, characterized in that a probe labeled with a third discrimination factor can hybridize with a part of a target nucleic acid located on the opposite side of the cleavage region to the important part side.

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